QC Report

	general
Report generated at	2025-02-21 23:26:55
Title	AdrenalGland
Description	ATAC-seq data from Liu et al. adrenal gland data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	98275020	148372008
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	96799143	146277744
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.5	98.6
Paired Reads	98275020	148372008
Paired Reads (QC-failed)	0	0
Read1	49137510	74186004
Read1 (QC-failed)	0	0
Read2	49137510	74186004
Read2 (QC-failed)	0	0
Properly Paired Reads	92921474	140562738
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	94.6	94.69999999999999
With itself	96591404	145940148
With itself (QC-failed)	0	0
Singletons	207739	337596
Singletons (QC-failed)	0	0
% Singleton	0.2	0.2
Diff. Chroms	108991	179857
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	37584934	59140850
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	28295037	45145531
Paired Optical Duplicate Reads	0	0
% Duplicate Reads	75.2829	76.3356

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	73310142	120830578
Rm = Number of Mitochondrial Reads	28752843	31204872
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.281716657610984	0.20524734198504363

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	17671986	27095634
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	17671986	27095634
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	17671986	27095634
Paired Reads (QC-failed)	0	0
Read1	8835993	13547817
Read1 (QC-failed)	0	0
Read2	8835993	13547817
Read2 (QC-failed)	0	0
Properly Paired Reads	17671986	27095634
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	17671986	27095634
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.727803445292057	0.7097313482990993
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	2.8570524457733693	2.6314509980747496
NFR / mono-nuc reads (QC pass)	True	True
NFR / mono-nuc reads (QC reason)	OK	OK
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.5404971461611615	0.5633351852922135
Fraction of Reads in blacklist regions	0.002277672696209696	0.0024203161291594063
Fraction of Reads in promoter regions	0.15713559302276495	0.17270365402780388
Fraction of Reads in enhancer regions	0.38301439351525063	0.39042286296013595

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	29348416	50236624
Distinct Fragments	8851836	13587085
Positions with Two Read	1860801	2571892
NRF = Distinct/Total	0.301612	0.270462
PBC1 = OneRead/Distinct	0.248319	0.219265
PBC2 = OneRead/TwoRead	1.181255	1.158356

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	100228	47792
N1	72116	31978
N2	88130	41224
Np	98916	48263
N optimal	100228	48263
N conservative	100228	47792
Optimal Set	rep1_vs_rep2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.013263779368353	1.0098552058921995
Self Consistency Ratio	1.2220589050973432	1.2891362811933205
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	146608	167556

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	171.0	182.0	508.0	312.0
50 percentile (median)	239.0	268.0	739.0	481.0
75 percentile	422.0	490.0	1005.0	771.0
Max size	2023.0	2317.0	2676.0	2676.0
Mean	347.6911219033068	388.22946358232474	782.3297142738744	580.7241090314084

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	18.365117628803656	18.997852749462098

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.15860412592945236	0.16536612401022868
Synthetic AUC	0.4885303342979942	0.4907629729562188
X-intercept	0.35298481497626466	0.2647561078028706
Synthetic X-intercept	2.7403761601626088e-64	1.0483506307287832e-99
Elbow Point	0.6935120716339598	0.7197789233310271
Synthetic Elbow Point	0.5091755206651345	0.5078636334415639
Synthetic JS Distance	0.3825571230454389	0.4211118149491841

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.2622629397737187	0.2980787974918764	0.29248876810011415	0.29568289151802896	0.29211819114367693	0.29466254929945906	0.2867303421535476	0.2745212477660195	0.2745352354702113

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.2529263561475906	0.2126307705313936	0.25411451896641357	0.25329079365845225

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.1975804833940245	0.15606768814778374	0.1944977925225887	0.19866293986591202

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates